This invention relates to an improved hemoglobin standard and to an improved total hemoglobin assay procedure utilizing the standard.
Ever since the early investigations of hemoglobin by Hoppe-Seyler, Gowers, Hufner and others in the 19th century, the importance of hemoglobin has been recognized, and simple diagnostic tests for hemoglobin concentration have been sought. In vitro measurements of hemoglobin are today among the most frequently performed diagnostic procedures and are important in the detection and management of anemia and other diseases associated with abnormal hemoglobin levels. Unfortunately, even now when millions of hemoglobin determinations are run annually and the accuracy of these tests is relied upon in choosing what therapy, if any, is to be utilized, there is no generally accepted, simple, sensitive and reproducible method for determining hemoglobin. See Federal Register 39, 9217 (Mar. 8, 1974).
Hemoglobin is also widely used in various types of research. A homogeneous, stable hemoglobin preparation of known quantity is thus a highly desirable article of commerce, and a simple colorimetric hemoglobin determination method would be highly valuable in the commercial manufacture of purified hemoglobin.
Mammalian hemoglobin is a chromoprotein in which a soluble basic protein molecule (globin), consisting of four polypeptide chains of similar molecular weight held together by non-convalent forces, is conjugated to four colored ferrous protoporphyrin (heme) molecules. The heme component is the same in all mammalian hemoglobins, but the globin component differs from species to species, from individual to individual of the same species, and even within the same individual.
Hemoglobin is capable of forming a number of derivatives attributable to the reactivity of its heme part, all of which are commonly referred to collectively as hemoglobin. This nomenclature will be used hereinafter, with the name ferrohemoglobin used for the parent molecule. In the circulating blood, hemoglobin's "respiratory function" is due to the reversible reaction of ferrohemoglobin with molecular oxygen to form oxyhemoglobin. These two forms of hemoglobin, therefore, constitute the active forms of hemoglobin in the circulating blood. Ferrohemoglobin also reacts reversibly with carbon monoxide to form carboxyhemoglobin and irreversibly with hydrogen sulfide to form sulfhemoglobin. Oxidizing agents, such as certain drugs or ferricyanide, oxidize the ferrous iron of ferrohemoglobin to the ferric state to form methemoglobin (ferrihemoglobin). Methemoglobin is reducible to ferrohemoglobin by reducing agents such as hydrosulfite, but it is unable to unite with molecular oxygen or carbon monoxide. Methemoglobin does form further derivatives by uniting with a number of anions, such as azide, cyanide and fluoride. All the common hemoglobins except sulfhemoglobin are interconvertible.
Ferrohemoglobin and its derivatives exhibit characteristic absorptioin spectra in the visible region of light, which provide a means for distinguishing them from one another both qualitatively and quantitatively. Generally, the spectral properties of ferrohemoglobin and its derivatives are not dependent on the nature of their globin components.
A number of chlorimetric methods have been described in the literature for the determination of the total hemoglobin concentration in whole blood. These methods initially pretreat a blood sample with a reagent, or diluting fluid, which converts all the hemoglobin in the blood sample into a suitable derivative. The color intensity of the diluted blood sample is measured in a colorimeter which has been calibrated with an appropriate color standard, either artificial or physiological. The use of a photoelectric colorimeter, rather than visual methods, is necessary to provide the degree of precision required in clinical tests. Although artificial color standards are regarded as stable, the estimation of hemoglobin using them is considered unreliable, largely because the light absorption properties of the standards do not exactly correspond with the light absorption properties of the hemoglobin derivatives. Therefore, colorimetric methods require the use of physiological standards derived by treating blood samples of known hemoglobin concentrations with the appropriate diluting fluid.
To avoid the discrepancies in hemoglobin estimation that arise from the heterogeneity of hemoglobin in blood (caused both by the genetic differences in the globin components and by the differences in relative proportions of ferrohemoglobin and its various derivatives), and to achieve uniformity in hemoglobin determinations, the National Academy of Sciences-National Research Council, U.S.A. (NRC), and the International Committee for Standardization in Haematology (ICSH) have both recommended the universal adoption of a "uniform" cyanmethemoglobin method and standard solution for colorimetric determination of hemoglobin. In brief, the method initially treats 0.02 ml of a blood sample with 5.0 ml of a diluting fluid to convert the hemoglobin in the sample into the cyanmethemoglobin derivative. The diluting fluid is an alkaline solution which contains 200 milligrams of K.sub.3 Fe(CN).sub.6 and 50 milligrams of KCN per liter. Color intensity is determined at 540 m.mu. with a photoelectric colorimeter. The colorimeter is calibrated with a cyanmethemoglobin standard solution which is of known concentration and which meets certain required specifications. The standard is produced by treatment of hemolyzed human blood with the standard diluting fluid in accordance with the recommended uniform method. The assignment of a universally accepted hemoglobin concentration to the standard solution by absolute spectrophotometry is made possible by arbitrarily assigning a molecular weight to hemoglobin of 64,458 (on the basis of the established molecular weight of hemoglobin-A), and by fixing the millimolar extinction coefficient of cyanmethemoglobin at 44.0. Therefore, the concentration of hemoglobin in the standard (mg./100 ml.) is given by equation (a): ##EQU1## Since the dilution factor of the procedure is 251, the corresponding gram percent of hemoglobin in the undiluted sample is given by Equation (b): ##EQU2## The recommendations of these two groups and discussions of the recommendations are contained, for example, in Cannan, Blood, 13, 1101 (1958); Cannan, Am. J. Clin. Pathol, 44, 207 (1965); Standardization in Hematology (ed. G. Astaldi et al.) Fondazione Carlo Erba, Milan, Italy 1970; Eilers, Am. J. Clin. Pathol., 47, 212 (1967); and Bibliotheca Haematologica, Vol 21 (ed. C. DeBoroviczeny), Basel/New York 1965.
The uniform cyanmethemoglobin method meets most of the requirements for a suitable colorimetric method. The diluting fluid converts to cyanmethemoglobin all forms of hemoglobin, except sulfhemoglobin, that are likely to be present in the circulation. Because the millimolar extinction coefficients of cyanmethemoglobin and sulfhemoglobin at 540 m.mu. are close, the presence of a small amount of sulfhemoglobin does not affect accuracy significantly.
The absorption curve of cyanmethemoglobin shows a flat maximum around 540 m.mu..
The diluting fluid may be prepared as a single reagent, and its pH and ionic strength do not influence the color intensity produced by the reaction, although they do determine the reaction time and the clarity of the final reaction mixture. A diluting fluid of low pH and ionic strength appears to favor both the rate of hemolysis and the rate of formation of cyanmethemoglobin, but is likely to cause turbidity in the final reaction mixture as a result of the partial solubility of plasma proteins and erythrocyte stromata, which require higher pH and ionic strength for solubilization. One diluting fluid in wide use is a low pH solution developed by Van Kampen and Zijlstra, which contains one hundred forty milligrams KH.sub.2 PO.sub.4 and 0.5 milliliters Sterox SE per liter in addition to the K.sub.3 Fe(CN).sub.6 and KCN. The solution has a low pH of about 7.2 and the Sterox SE, a non-ionic surfactant, reduces turbidity to an acceptable level. A second widely used diluting fluid is based on a recipe by Drabkin and includes one gram of NaHCO.sub.3 per liter in addition to the K.sub.3 Fe(CN).sub.6 and KCN, to maintain a relatively high pH of about 8.6. Both diluting fluids suffer from deterioration over a period of time. At low temperatures (refrigerator), the K.sub.3 Fe(CN).sub.6 and KCN tend to form K.sub.4 Fe(CN).sub.6 and KCNO, with an attendant fading of the yellow color of the solution. At room temperature the solutions are susceptible to contamination by microorganisms. The solutions are particularly likely to undergo changes due to temperature variations during shipping, even if they have been sterilized and are shipped in hard borosilicate glass containers. Weatherburn and Logan, Clin. Chim. Acta., 9, 581 (1964) have suggested the preparation and distribution of a dry mixture of Drabkin's ingredients. The dry mixture is temperature insensitive and may be shipped without danger of decomposition. The Van Kampen-Zijlstra solution reacts faster with a sample but is not amenable to shipment as a dry mixture. Attempts to ship it as a concentrate have failed because of rapid decomposition.
The greatest problem with the NRC-ICSH recommended uniform cyanmethemoglobin colorimetric method lies in the standard solution required for calibrating the colorimeter. An approved cyanmethemoglobin standard solution is prepared from red cell hemolysate free from erythrocyte stromata, by diluting the hemolysate with the same diluting fluid which is to be used for treating blood samples. To assure that the standard solution is suitable, it must carry the seal of certification of competent authorities recognized by NCR-ICSH and must meet specifications with regard to its hemoglobin concentration, purity, and turbidity. Concentration, checked spectrophotometrically, should be between 59.77 and 79.69 mg. hemoglobin/100 ml. (OD(1 cm. path)=0.408-0.544), which is equivalent to between 15 and 20 gram percent hemoglobin in whole blood, diluted 1:251. The concentration must not change more than .+-.2% during the stated life of the standard. The purity of the standard is checked by inspecting the shape of its absorption curve between 450 and 700 m.mu. for consistency with a curve derived from a pure cyanmethemoglobin solution. Purity is also checked spectrophotometrically by determining the ratio of optical density at 540 m.mu. to optical density at 504 m.mu.. This ratio should be between 1.58 and 1.62 because those standard solutions with the highest ratios (i.e. those in this range) appear to be the most stable. The turbidity of the standard is checked spectrophotometrically by measuring its OD.sub.750 (1 cm. path), the value of which should be less than or equal to 0.002.
If stipulated criteria for purity and clarity were always met by carefully prepared cyanmethemoglobin standard solutions, and if meeting these criteria guaranteed the suitability of the cyanmethemoglobin standard for calibrating or recalibrating a colorimeter many days and miles removed from the time and place the tests were met, the NRC-ICSH recommendations for a uniform cyanmethemoglobin method would probably be widely accepted as a relatively simple, sensitive, and reliable method for determining total hemoglobin. However, the criteria are not always met and the stability of even the most carefully prepared standard solutions is open to serious question.
Some of the problems with meeting the criteria are set out, for example, in a paper by Moran entitled "Challenge to the Criteria" in Standardization in Hemotology (ed. G. Astaldi et al.) Fondazione Carlo Erba, Milan, Italy 1970. Briefly, although changes with time in the OD.sub.540 /OD.sub.504 value of a particular standard solution probably indicate deterioration, the measured value of this ratio for any given freshly prepared standard rarely falls within the prescribed range. This is true whether the standard is prepared from crystalline hemoglobin preparations or from red cell hemolysate and in spite of the fact that freshly prepared cyanmethemoglobin standard solutions obtained from purified hemoglobin preparations consistently produce absorption curves that are characteristic of cyanmethemoglobin. The usefulness of the OD.sub.750 measurement as an indication of turbidity has also been questioned by Moran and others.
Although the cyanmethemoglobin standard solution has sometimes been reported as stable in storage, if maintained in the dark under sterile conditions at 0.degree.-4.degree.C, there is considerable evidence that the stability of the solution is at best unpredictable, particularly during shipment. See, for example, Cannan, Blood, 13, 1101 (1958); Ressler et al. J. Lab. & Clin. Med., 54, 304 (1959); and Von Klein-Wisenberg & Clotten, in Bibliotheca Haematologica, Vol. 21, pp. 79-95. These authors suggest that the instability is due to the microorganisms or to impurities in the standard, and that the solutions should be made more carefully, although they do not suggest exactly what care is necessary. The instability of the diluting fluid portion of the standard affects the optical density of the standard at 540 m.mu.. More importantly, in an alkaline solution containing K.sub.3 Fe(CN).sub.6 the stability of the hemoglobin itself is doubtful. The lability of proteins in dilute solution, and in particular of human hemoglobin, has frequently been commented upon, for example by Cannan, supra, and by Itano in Advances in Protein Chemistry, Vol. 12 at 212, 222. Work by Riggs and Wolbach, J. Gen. Physiol., 39, 585, 600 (1956), and by Mirsky and Anson, J. Gen. Physiol., 19, 439 (1936), indicates the oxidation of the SH groups of hemoglobin in alkaline solutions of hemoglobin similar to the proposed standard, and therefore suggests that the cyanmethemoglobin standard solution is liable to undergo deterioration in storage due to intermolecular disulfide bond formation.